Vancomycin

As stated above, parallel samples were prepared for BAM 4a analysis using the same inoculum and procedures as for the flow cytometry procedure. The samples were then processed, using a modification of the standard regulatory procedure. Since we only used a 25 g sample of spinach per bag, instead of a 200 g sample amount (typically specified for composite samples), a proportionally lower amount of sterile PBS was added (i.e., 25 mL) to each sample before they were placed on a shaker-incubator for 5 min. 20 mL of 2× modified buffered peptone water pyruvate (mBPWP, Remel, Labsource, Romeoville, IL, United States) was added to each before placing them back into the incubator at 37°C for 5 h. At this point the BAM4a specifies addition of inhibitors to which background microflora are typically more sensitive than the target E. coli O157:H7. We added 333 μL of an ACV cocktail (Acriflavine, Cefsulodin, Vancomycin) containing Acriflavine and Cefsulodin at 7.5 × 10^-4 g/mL each and Vancomycin at 6.0 × 10^-4 g/mL, all from MP Biomedicals, LLC, Solon, OH, United States. The volume added was adjusted proportionally for the smaller 45 mL suspension volume for single 25 g spinach samples. All subsequent processing for the regulatory samples was the same as outlined in the BAM manual (Feng et al., 2011 ).

Processing of rectal swabs and blood culture have been described in Figure 1 and in supplementary methods. Briefly, rectal swabs were plated on vancomycin (10 mg/L) (MP Biomedicals, California, USA) supplemented chrome agar (CA) (BD BBL, MD, USA) and incubated (37°C, 18–24 h). Cultures from the primary inoculum of each plate and cultured isolates from neonatal blood were screened for mcr genes by polymerase chain reaction (PCR) as described previously [18 (link)]. Sample positive for mcr genes were further enriched in Enterobacteriaceae Enrichment (EE) Mossel broth (37°C, 18–24 h) (BD BBL) and plated on to CA supplemented with/without colistin sulphate (2 mg/L) (MP Biomedicals).
Colonies with different colours were picked from plates with/ without colistin, and again screened for mcr genes. Amplified PCR products were Sanger sequenced and stocked for further analysis [12 (link)]. Any mcr-negative colony of similar colour as the mcr-positive ones were collected from CA plates (without colistin) to check clonality with mcr-positive colonies.